A protein is synthesized (transcription and translation) based on the nucleotide information of DNA, the entity of a gene. It is known that activities and functions in most proteins are regulated by further modification after translation. Phosphorylation is one of the most investigated posttranslational modifications of proteins. Many of the oncogene family proteins, such as c-Src and c-Raf, which manage intracellular signal transduction are regulated by phosphorylation and dephosphorylation, and these intracellular signal transductions themselves are conduced by a sequence of phosphorylation and dephosphorylation (Morrison, D. K., Kaplan, D. R. et al. (1989) Cell 58, 649-657; Howe, L. R., Leevers, S. J. et al. (1992) Cell 71, 335-342; Kolch, W., Heidecker, G. et al. (1993) Nature 364, 249-252; Dent, P., Jelinek, T. et al. (1995) Science 268, 1902-1906). Even in the nucleus of a cell, many transcription factors and their regulatory proteins are known to be minutely regulated by phosphorylation and dephosphorylation (Hill, C. S., Marais, R. et al. (1993) Cell 73, 395-406; Sanchez, I., Hyghes, R. T. et al. (1994) Nature 372, 794-798; Akoulitchev, J., Makela, T. P. et al. (1995) Nature 377, 447-560; Weinberg, R. A. (1995) Cell 81, 323-330). As another posttranslational modification, many extracellular proteins and cell-surface proteins, such as receptors, have been reported to be subjected to glycosylation, such as the addition of a glycosyl group (Guan, J. L., Machamer, C. E. and Rose, J. K. (1985) Cell 42, 489-496; Sairam, M. R. and Bhargavi, G. N. (1985) Science 229, 65-67; Diamond, M. S., Staunton, D. E et al. (1991) Cell 65, 961-971; Entwistle, J., Hall, C. L. and Turley, E. A. (1996) J. Cell. Biochem. 61, 569-577). Such glycosylations are proposed to have an important role in the formation of the higher-order structure of the extracellular matrix and cell-surface receptors, and intercellular recognition. GTP binding protein family, such as Ras, has been known to be modified with lipids by farnesylation and addition of palmitic acid (Willumsen, B. M., Christensen A. et al. (1984) Nature 310, 583-586; Buss, J. E., Solski, P. A. et al. (1989) Science 24, 1600-1603; Lowy, D. R. and Willumsen, B. M. (1989) Nature 341, 384-385; Vogt, A., Qian, Y et al. (1995) J. Biol. Chem. 270, 660-664). These modifications are thought to be important for the localization of proteins into the cell membrane and the interaction with other proteins.
Acetylation has been reported as a posttranslational modification only in histone. Histone is a basic protein binding to DNA and forms a nucleosome, the basic structural unit of chromatin. It was reported that this protein is highly acetylated at activated chromatin sites, where mRNA is actively transcribed, while the acetylation level is low at inactivated chromatin sites (Hebbes, T. R., Throne, A. W. and Crane-Robinson, C. (1988) EMBO J. 7, 1395-1402; Wolffe, A. P. (1996) Science 272, 371-372). As enzymes which transfer an acetyl group to histone from acetyl CoA in mammalian cells (histone acetyltransferase: HAT), five kinds; GCN5 (Kuo, M.-H., Brownell, J. E. et al. (1996) Nature 383, 269-272; Brownell, J. E., and Allis, C. D. (1996) Curr. Opin. Genet. Dev. 6, 176-184; Candau, R., Zhou, JX., Allis, C. D. and Beregr, S. L. 1997) EMBO J. 16, 555-565), P/CAF (Ogryzko, V. V., Sciltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959) p300/CBP (Bannister, A. J., and Kouzarides. T. (1996) Nature 384, 641-643; Yang, X.-J., Ogryzko, V. V. et al. (1996) Nature 382, 319-382), TAFII250 (Mizzen, C. A., Yang, X.-Y. et al. (1996) Cell 87, 1261-1270), Tip60 (Kimura, A., Yamamoto, Y., Horikosi, M., Institute of Molecular and Cellular Biosciences, Laboratory of Developmental Biology, the University of Tokyo, Analysis of a novel histone acetyltransferase Tip60 family, presented at The Twentieth Annual Meeting of Japanese Society of Molecular Biology, Dec. 17, 1997) have been reported. As enzymes which deacetylate histone (histone deacetylase), three genes; HDAC1/RPD3 (Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) Science 272, 408-411; Rundlett, S. E., Carmen, A. A. et al. (1996) Proc. Natl. Acad. Sci. USA. 93, 14503-14508), HDAC2/YY-1BP (Yang, W.-M., Inouye, C., zeng, Y. Y., Bearss, D., and Seto, E. (1996) Proc. Natl. Acad. Sci. USA. 93, 12845-12850: Lusser, A., Brosch, G. et al. (1997) Science277, 88-91), HDAC3 (Yang, W.-M., Yao, Y.-L., Sun, J.-M., Davie, J. R., and Seto, E. (1997) J. Biol. Chem. 272, 28001-28007), have been reported.
Recently, it was reported that p300/CBP reported as a HAT, acetylates not only histone, but also p53, enhancing p53 activity (Scolinick, D. M., Chehab, N. H. et al. (1997) Cancer Res 57, 3693-3696; Gu, W., Shi, X.-L., and Roeder, R. G. (1997) Nature 387, 819-823; Lill, N. L., Grossman S. R. et al. (1997) Nature 387, 823-827; Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606). p53 had been identified as an intranuclear protein specifically and highly expressed in cancerous cells, and was thought to be an oncogene by experiments such as transduction experiments using p53 gene isolated from cancerous cells. However, p53 gene isolated from cancerous cells was identified to be a mutant, and it was found that the normal p53 gene is in fact a cancer suppressor gene because the normal p53 gene shows phenotypes of inhibiting cellular proliferation, arresting the cell cycle, and inducing cell death, etc. It is proposed that expression of p53 is induced by DNA damage and such, and functions as a transcriptional factor by binding to a specific sequence of DNA, illustrating the function as a cancer suppressor gene. Binding ability of p53 to a specific DNA is enhanced by acetylation, and as a result, transcriptional activation is also elevated. It has been also reported that transcriptional activity of p53 is controlled by phosphorylation. The report that acetylation strongly induces enhancement of transcriptional activity of p53 implies not only the existence of a novel regulatory mechanism, but also the possibility that acetylation, like phosphorylation, is involved in the control of protein function not only in histone but in cells in general. Thus, enzymes relating to phosphorylation, dephosphorylation and lipid modifications, and their substrate-proteins have received wide attention recently as targets in development of novel drugs such as immune inhibitors and anticancer agents. Screening for inhibitors against these enzymes are underway.
Considering the circumstances, acetylation, deacetylation and their relating proteins are expected to be new targets in drug development in the future. So far, drugs such as sodium butyrate, trichostatin A, and trapoxin, have been reported as inhibitors for histone deacetylase. These inhibitors have been originally identified as antifungal agents or morphological normalization substances for v-sis-transformant cells, causing arrest of the cell cycle and induction of cell differentiation (Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) Science 272, 408-411; Yoshida, M., Kijima, M., Akita, M., and Beppu, T. (1990) J. Biol. Chem. 265, 17174-17179; Kijima, M., Yoshida, M. and et al. (1993) J. Biol. Chem. 268, 22429-22435; Chen, W, Y., Bailey, E. C. et al. (1997) Proc Natl. Acad. Sci. USA, 94, 5798-5803; Medina, V., Edmonds, B. et al. (1997) Cancer Res. 57; 3697-3707). Later studies demonstrated that the target of these drugs is histone deacetylase. These kinds of inhibitors are expected to function as anticancer drugs and antimicrobial agents, and screening of histone deacetylase inhibitors as a search for substances comprising a similar function is expected to be carried out in the future.
The methods known for measuring the acetyltransferase and deacetylase activities are, however, very cumbersome. Specifically, to measure acetyltransferase activity, acetyltransferase and radiolabeled acetyl CoA are added to histone purified from cells or a synthetic peptide substrate to execute the acetyl group-transferring reaction. Each reaction solution is then transferred onto a filter and washed to measure enzyme activity using a liquid scintillation counter (Bannister, A. J., and Kouzarides. T. (1996) Nature 384, 641-643; Mizzen, C. A., Yang, X.-Y et al. (1996) Cell 87, 1261-1270; Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606; Brownell, J. E. and Allis, C. D. (1995) Proc. Natl. Acad. Sci. USA 92, 6364-6368). To measure the deacetylase activity, radiolabeled acetic acid is added into a medium of cultured cells to metabolically radiolabel cellular histone. Histone is purified from the cells, and deacetylase is reacted to the histone for the deacetylation reaction. After the completion of the reaction, radiolabeled acetyl group which is released from histone is isolated and extracted with ethyl acetate to measure the enzyme activity by a liquid scintillation counter (Laherty, C. D., Yang, W.-M. et al. (1997) Cell 89, 349-356; Hassig, C., Fleischer, T. C. et al. (1997) Cell 89, 341-347; Hendzel, M. J., Delcuve, G. P. and Davie,-J. R. (1991) J. Bio. Chem. 32, 21936-21942).
These measurement systems are so cumbersome that assaying many samples under numerous conditions is difficult. Therefore, a simple and convenient screening system for new drug development and such was desired.